Determining the minimum ripening time of artisanal Minas cheese,a traditional Brazilian cheese

José M. Martins1, Éder Galinari2, Natan J. Pimentel-Filho2, José I. Ribeiro Jr3,Mauro M. Furtado4, Célia L.L.F. Ferreira4

1Instituto Federal de Educação, Ciência e Tecnologia, Rio Pomba, MG, Brazil.2Departamento de Microbiologia, Universidade Federal de Viçosa, Viçosa, MG, Brazil.

3Departamento de Estatística, Universidade Federal de Viçosa, Viçosa, MG, Brazil.4Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Viçosa, MG, Brazil.

Submitted: September 17, 2013; Approved: June 6, 2014.

Abstract

Physical, physicochemical, and microbiological changes were monitored in 256 samples of artisanalMinas cheese from eight producers from Serro region (Minas Gerais, Brazil) for 64 days of ripeningto determine the minimum ripening time for the cheese to reach the safe microbiological limits estab-lished by Brazilian legislation. The cheeses were produced between dry season (April-September)and rainy season (October-March); 128 cheeses were ripened at room temperature (25 � 4 °C), and128 were ripened under refrigeration (8 � 1 °C), as a control. No Listeria monocytogenes was found,but one cheese under refrigeration had Salmonella at first 15 days of ripening. However, after22 days, the pathogen was not detected. Seventeen days was the minimum ripening time at room tem-perature to reduce at safe limits of total coliforms > 1000 cfu.g-1), Escherichia coli and Staphylococ-

cus aureus (> 100 cfu.g-1) in both periods of manufacture. Otherwise under refrigeration, as expected,the minimum ripening time was longer, 33 days in the dry season and 63 days in the rainy season. Tosum up, we suggest that the ripening of artisanal Minas cheese be done at room temperature, sincethis condition shortens the time needed to reach the microbiological quality that falls within the safetyparameters required by Brazilian law, and at the same time maintain the appearance and flavor char-acteristics of this traditional cheese.

Key words: ripening, artisanal Minas cheese, Serro, pathogen, lactic acid bacteria.

Introduction

Artisanal cheeses are produced in many countries andhave specific properties depending on the region wherethey are made. In Brazil, the State of Minas Gerais is knownas the major cheeses producer, and the Serro region isamong the main production regions of the artisanal Minascheeses. The artisanal Minas cheese is made from rawcow’s milk, mainly involving family labor, which, in mostcases, does not have sufficient resources to adapt their pro-duction facilities to improve the product quality. The mar-keting of artisanal food products in a safe and sustainableway is one of the main challenges faced by family farmersin Brazil; it still receives little attention from government

agencies responsible for its development. In an attempt toimprove the quality of artisanal Minas cheese, the govern-ment has created laws that establish the physicochemicaland microbiological parameters to which these cheesesmust adhere (Minas Gerais, 2002, 2008). However, to becommercialized throughout the country, the cheese mustmeet the standards required by federal law (Brasil, 2000)that stipulate a minimum ripening time of 60 days forcheeses made from raw milk to ensure safety since the pro-cess of ripening can contribute significantly to the reduc-tion of pathogens in such products.

Ripening is a complex phenomenon that involvesphysical, physicochemical, and microbiological changes

Brazilian Journal of Microbiology 46, 1, 219-230 (2015) Copyright © 2015, Sociedade Brasileira de MicrobiologiaISSN 1678-4405 www.sbmicrobiologia.org.brDOI: http://dx.doi.org/10.1590/S1517-838246120131003

Send correspondence to J.M. Martins. Instituto Federal de Educação, Ciência e Tecnologia, Campus Rio Pomba. Avenida Dr. José Sebastião da Paixãos/n, Lindo Vale, 36180-000 Rio Pomba, MG, Brazil. E-mail: jose.manoel@ifsudestemg.edu.br.

Research Paper

that occur in cheese under the influence of environmentalfactors and enzymatic and organic acids produced, mostlyby natural milk microbiota (Dolci et al., 2010; Sihufe et al.,2010). This microbiota - composed mostly by lactic acidbacteria (LAB) - are more complex than industrial startercultures added to pasteurized milk during cheese makingand play a strong role in lipolysis and proteolysis, resultingin compounds responsible for flavor and texture character-istics. Most LAB release substances that control undesir-able microorganisms counts in cheese (Cabezas et al.,2005; González et al., 2007).

Since these cheeses are microbiologically safe, newperspectives may be established in order to characterizethem in each producing region, with the adoption of quanti-tative descriptive analysis and especially certification of or-igin, which can guarantee recognition for making a genuinetraditional cheese to the cheese maker. This adds value tothe product and protects it from the clandestinity. Con-sidering the excessively long ripening time established byBrazilian law and the intention of characterizing artisanalMinas cheese, this research aimed to study the physical,physicochemical, and microbiological changes that occurin this cheese during the ripening, suggesting a minimumripening time at room temperature that ensures microbio-logical safety and at the same time maintains the traditionalsensory characteristics. These cheeses are traditionally rip-ened at room temperature, but due to legislation require-ments that establish packaging and refrigeration duringripening, we included refrigeration as a control treatment.This is the first study that characterizes a Brazilian artisanalcheese and provides relevant information to support cheesemakers in obtaining protected geographical indication sta-tus.

Materials and Methods

Sampling and conditions of cheese ripening

We randomly selected eight producers who makeartisanal Minas cheese (and are affiliated with the FarmersCooperative of Serro) at different levels of adequacy to leg-islation requirements for good agricultural practices (MinasGerais, 2002).

Aliquots of 100 mL of water, endogenous culture(EC), and milk used to manufacture cheeses were collectedfrom each farm. After two days of production, at the end ofsalting, the cheeses were collected from the farms of origin.Water samples, EC, milk, and cheeses with two days of pro-duction were microbiologically analyzed as described attopic 2.4. A total of 256 cheese samples were collected intwo seasons, dry season (April-September) and rainy sea-son (October-March).

The average temperature and relative humidity (RH)storage of cheeses during the ripening were: i) dry season:ripening at room temperature (23 � 2 °C and 62% RH) andripening in a cold chamber (8 � 1 °C and 75% RH); and ii)

rainy season: ripening at room temperature (27 � 2 °C and68% RH) and ripening in cold chamber at (8 � 1 °C and77% RH). The cheeses ripened at room temperature werestored without packaging in a metal cabinet, with mesh oneither side for ventilation and shelves lined with aluminumfoil. The cheeses were washed with potable water andturned every two days. The samples ripened under refriger-ation were vacuum-packed and stored on shelves in a coldchamber. Physical, physicochemical, and microbiologicalanalysis were performed on the cheeses at 8, 15, 22, 29, 36,50, and 64 days of ripening.

Physical analysis

The weight of the cheeses was determined using adigital scale (Filizola, Model BP15, Brazil). Height and di-ameter were measured with a ruler.

Physicochemical analysis

The cheeses were analyzed for acidity (AOAC,1990), fat (IDF, 1969), moisture (IDF, 1982) and chlorides(IDF, 1988). The pH was measured using a portable digi-tal pH meter (Tecnal, Model 102) using a specific electrodefor cheese. Fat in dry matter (FDM) was determined indi-rectly as follows: %FDM = (%F/%DM) x 100, in which %Fis the fat content of the sample and %DM is the content oftotal solids in the sample (AOAC, 1995). The water activity(Aw) was determined using an Aqualab device (DecagonModel CX2T). Total nitrogen (TN), pH 4.6 soluble nitro-gen (pH 4.6 SN), and soluble nitrogen in 12% (v/v) trichlo-racetic acid (TCA-SN) were determined by the Kjeldahlmethod (IDF, 1993). The total protein (TP) content was de-termined indirectly by multiplying the percentage of TN bya factor of 6.38, indicated for protein derived from milk(IDF, 1993). Ripening extension index (REI; Eq. (1)) andripening depth index (RDI; Eq. (2)) were calculated:

REI = (pH 4.6 SN/TN) x 100 (1)

RDI = (TCA-SN/TN) x 100 (2)

Microbiological analysis

For microbiological analysis, 25 g of each cheesewere sampled by removing a slice from the center to the pe-riphery of the cheese, in different areas, using a sterilecheese trier. Each sample was homogenized with 225 mLof 0.12% (w/v) sterile phosphate buffer solution in a Sto-macher 400 Bagmixer® (Model VW, France) for 2 min atlow speed. Serial decimal dilutions were performed beforeplating in depth. The total mesophilic aerobe count was de-termined in samples of milk according to the AmericanPublic Heath Association (2001). Total coliform bacteriaand Escherichia coli were enumerated in samples of water,milk, EC, and cheese, and Staphylococcus aureus wereenumerated in milk samples, EC, and cheese using specificPetrifilm® plates (3M, Minnesota, USA). Salmonella spp.and Listeria monocytogenes were evaluated in the cheeses

220 Martins et al.

beginning at two days of ripening using Reveal® kits (Neo-gen, Miami, USA).

Staphylococcal enterotoxins analysis

We evaluated the presence of staphylococcal entero-toxins in samples of artisanal Minas cheese, in the dry andrainy seasons, using the qualitative immunoenzimatic testVIDAS® Staph Enterotoxin (BioMérieux, Marcy-l’Étoile,France) that allows simultaneous detection of seven differ-ent enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED,SEE).

Statistical analysis

The research was performed on scheme of sub-divided parcels, in which the parcels consist of ripeningconditions (room temperature and refrigeration) in a ran-domized block design with eight replicates (cheese mak-ers). The sub-parcels consisted of periods of manufacture(dry and rainy seasons), and the sub-sub-parcels consistedof ripening time (2, 8, 15, 22, 29, 36, 50, and 64 days).Analysis of the cheeses two days after manufacture wascarried out only for the determination of microbial counts,without the interference of the ripening period.

Data were subjected to variance analysis (ANOVA)analysis to determine the main effects and interactions ofsecond and third orders among the three factors studied (pe-riod of manufacture, ripening condition, and ripeningtime). Depending on the significance of the interactions, weperformed an F-test to compare means of two ripening con-ditions and the two periods of manufacture at the 5% signif-icance level. We conducted regression analysis on theripening time for each combination of the ripening condi-tions and period of manufacture, the coefficients of whichwere subjected to a Student’s t-test up to the 5% signifi-cance level. All statistical analysis were performed in soft-ware SAEG 9.1 (UFV, Viçosa, Minas Gerais, Brazil).

Results and Discussion

Physical characteristics of cheeses

The ripening at room temperature reduced (p < 0.05)the average weight, height, and diameter in both periods ofmanufacture, while the refrigeration caused no effect(p > 0.05) (Figure 1). The explanation for this phenomenonis the loss of moisture from the cheeses. Moreover, the re-frigerated cheeses retained their moisture content due to thepresence of a plastic film that hinders moisture exchangewith the environment. The diameter was the parameter thatshowed the greatest homogeneity among the samples, pos-sibly due to use of moulds with a similar diameter amongthe cheese makers. Because artisanal Minas cheese is notpart of the certification systems that establish criteria forstandardization, starting with their own sizing, these resultsdemonstrate the heterogeneity among producers.

Physicochemical characteristics of cheeses

The lactic acid concentration was not affected(p > 0.05) by temperature and ripening time. However, theperiod of manufacture affected (p < 0.05) almost all param-eters, except for Aw, sodium chloride, moisture, andweight. Fat in dry matter was affected (p < 0.05) by the con-dition of ripening and the period of manufacture. Cheesesripened at room temperature showed higher FDM means(p < 0.05) than the cheeses ripened under refrigeration(53.29a and 49.90b, respectively). When the cheeses weremanufactured in the dry season, they also had higher FDMmeans (p < 0.05) than those made during rainy season(53.45a and 49.74b, respectively). The cheeses can be clas-sified based on the content of FDM, in accordance with thestandards established by federal legislation (Brasil, 1996).Thus, the cheeses in this study, regardless of ripening timeor temperature, were classified as fat (45 to 59.9% FDM).

Interactions (p < 0.05) were observed between thefactors temperature and period of manufacture for TCA-SNand RDI. In both periods of manufacture, the RDI ofcheeses was higher (p < 0.05) at room temperature, and inthis same condition of ripening, RDI values were higher(p < 0.05) in cheeses during the rainy season (Table 1).These data indicate that higher temperatures accelerate theprocess of cheese ripening, especially those made from rawmilk, due to the greater presence of aminopeptidases andcell lysis, with the release of endo- and exopeptidases(Gorostiza et al., 2004; Grappin and Beuvier, 1998).

The temperature and ripening time factors affected(p < 0.05) various physicochemical parameters, except forFDM, lactic acid, and REI. In general, all cheeses that wereripened at room temperature showed lower (p < 0.05) meanAw and moisture over much of the entire ripening time,while sodium chloride, TN, TP, and pH 4.6 SN had higher(p < 0.05) means in the same conditions. These results canbe explained by the water loss of cheeses due to elevatedtemperatures (23-27°C) and low RH (62-68%).

An interaction (p < 0.05) between the ripening timeand period of manufacture occurred for Aw, sodium chlo-ride, pH, lactic acid, pH 4.6 SN, REI, TCA-SN and RDI.The average sodium chloride means were higher (p < 0.05)during the dry season, but only at 29 days and 36 days ofripening. Dry salting used by cheese makers in Serro doesnot allow for the standardization of the salt content in

Minimum ripening time of an artisanal cheese 221

Table 1 - Averages of RDI artisanal Minas cheese of the Serro region, ac-cording to the period of manufacture and condition of ripening.

Period of manufacture Ripening temperature

Room Refrigeration

Rainy season 10.05 a A 7.20 a B

Dry season 8.50 b A 6.97 a B

Means followed by same lowercase letter in the columns, and uppercaseletter in the lines, do not differ based on the results of an F-test (p > 0.05).

cheese. The rainy season had higher (p < 0.05) mean Aw

than the dry season only at 36 days of ripening. This resultcorrelates with the sodium chloride measurements, inwhich it was inversely proportional to Aw. The pH meanswere higher (p < 0.05) during the rainy season throughoutthe entire ripening time, whereas the lactic acid means werehigher (p < 0.05) during the dry season, beginning with thethird week of ripening. In production areas of artisanalMinas cheese, it is common to use a greater amount of ECduring the dry season, offsetting the adverse effects oflower temperatures on the fermentation process, which ex-plains the differences between the two periods of manufac-ture for pH and lactic acid.

The averages of pH 4.6 SN over the entire ripeningperiod and REI from 29 days on were higher (p < 0.05) dur-ing ripening in the rainy season, possibly due to increasedtemperature in this period, which favors the action of coag-ulating enzymes in the breakdown of casein, forming largepeptides from intermediate size. The RDI had higher means(p < 0.05) during the rainy season, but only after 50 days ofripening.

The interaction among ripening time, ripening condi-tion, and period of manufacture occurred (p < 0.05) only forfat and TCA-SN. Cheeses ripened at room temperatureshowed higher (p < 0.05) mean of fat contents than cheesesrefrigerated during the entire ripening time in both periodsof manufacture. The loss of moisture from the cheese wasfacilitated by environmental conditions (higher tempera-tures and low RH) that increased the total solids, and conse-quently the fat content, during ripening. Furthermore, thecheeses made in the dry season had higher (p < 0.05) meanof fat contents than those made during the rainy season, butonly after 22 days for cheeses ripened at room temperature,and after 50 days for refrigerated cheese. The fat content ofmilk varies depending on the season, the type of animalfeed used, genetic factors such as animal size, weight, andproduction, animal age, and the period of lactation (Bell et

al., 2006; Shingfield et al., 2006; Stanton et al., 1997).Higher TCA-SN (p < 0.05) means were found in cheesesripened at room temperature, in both periods of manufac-ture throughout virtually the entire ripening time. However,after 22 days, the rainy season had the highest (p < 0.05) av-erage of TCA-SN when the cheeses were ripened at roomtemperature. Under refrigeration, time did not affect meanTCA-SN (p < 0.05) in both periods of manufacture.

We observed gradual increases (p < 0.05) in fat (Fig-ure 1), TN, and pH (Figure 2) in both periods of manufac-ture when the cheeses were ripened at room temperature,which was not verified in the refrigerated cheeses(p < 0.05), as occurred in other artisanal cheeses (Caridi et

al., 2003a; Cichoski et al., 2002; Raphaelides et al., 2005).The increases (p < 0.05) in fat and TN of the ripenedcheeses at room temperature are directly related to moistureloss of cheese during the ripening time, which contributesto the concentration of total solids, while in the refrigerated

cheeses, these parameters remain practically unchanged.The increase (p < 0.05) in pH during ripening at room tem-perature may be due to protein degradation from the activ-ity of native milk protease (plasmin) and the proteasepresent in the EC, with the formation of alkaline nitroge-nous compounds.

Means of lactic acid concentrations (Figure 2) werenot affected (p < 0.05) by ripening time in both periods ofmanufacture and in the two ripening conditions. In the pres-ence of calcium, lactic acid can be converted to calcium lac-tate, maintaining its equilibrium (Chevanan et al., 2006).

Means of pH 4.6 SN (Figure 2) and TCA-SN (Fig-ure 3) concentrations increased (p < 0.05) throughout ripen-ing in both periods of manufacture and ripening conditions,especially at room temperature. Higher temperatures favorprimary proteolysis, represented by pH 4.6 SN, which isformed by the breakdown of proteins during cheese ripen-ing, primarily involving the conversion of large casein pep-tides, mainly due to residual action of the coagulant with�-S1, �-, and �-caseins (Bertolino et al., 2011). The highestaverage of TCA-SN (p < 0.05) at room temperature can beexplained by the presence of low molecular weight sub-stances (amino acids, oligopeptides, amines, and others),also responsible for the sensory characteristics of cheeses,which accumulate during the ripening time due especiallyto the proteolytic action of microbial enzymes (Prieto et al.,2004).

Throughout ripening in both periods of manufacture,the moisture and Aw (Figure 3) decreased (p < 0.05) incheeses stored at room temperature, while the sodium chlo-ride content (Figure 3) increased (p < 0.05). Moreover,none of these parameters changed (p < 0.05) throughout theripening time in refrigerated cheeses.

Federal law (Brasil, 1996) classifies cheeses by theirmoisture content, so the cheeses in this research that wereripened under refrigeration were classified as high moisture(46 to 54.9%). In contrast, ripened cheeses at room temper-ature were classified as medium moisture (36 to 45.9%)when they were at 8 to 17 days of ripening and low moisture(< 36%) after 18 days of ripening. During ripening, highertemperatures accelerate decrease in the moisture content ofcheeses and, consequently, reduce Aw with increased totalsolids, such as sodium chloride (Cabezas et al., 2005; Öneret al., 2006; Prieto et al., 2004). In general, sodium chlo-ride, Aw, and redox potential, as well as other physico-chemical factors such as lactic acid and pH, can directlyinfluence the microbiota present in cheese, contributing totheir microbiological safety.

Microbiological analysis of water, endogenousculture, and milk

Of water samples, 56.25% were found to be above thestandards required by legislation for coliforms (absence in100 mL) and were up 31.25% for E. coli (data not shown).The coliform counts were slightly higher during the rainy

222 Martins et al.

Minimum ripening time of an artisanal cheese 223

Figure 1 - Estimates of weight, height, diameter, and fat content in samples of artisanal Minas cheese from the region of Serro, as a function of the ripen-ing time in days (D). Room - ripening at room temperature, Ref - ripening under refrigeration. *Significant by Student’s t-test (p < 0.05).

224 Martins et al.

Figure 2 - Estimates of total nitrogen, pH, lactic acid, and pH 4.6 SN in samples of artisanal Minas cheese from the region of Serro, as a function of the rip-ening time in days (D). Room - ripening at room temperature, Ref - ripening under refrigeration. *Significant by Student’s t-test (p < 0.05).

Minimum ripening time of an artisanal cheese 225

Figure 3 - Estimates of TCA-SN, moisture, Aw, and NaCl in samples of artisanal Minas cheese from the region of Serro, as a function of the ripening timein days (D). Room - ripening at room temperature, Ref - ripening under refrigeration. *Significant by Student’s t-test (p < 0.05).

season. The presence of E. coli indicates recent fecal con-tamination, and thus, that water cannot be used. The adop-tion of an appropriate treatment to ensure the physico-chemical and microbiological safety of water, such as theuse of efficient filters and chlorination systems, is very im-portant in artisanal Minas cheese production, since it is es-sential for cleaning facilities and equipment. Table 2 pres-ents the mean microbial counts of milk and EC used in themanufacture of artisanal Minas cheese from the region ofSerro. The EC, which is collected at the end of syneresis ofthe cheeses, presented in the two periods of manufacturehigh counts of coliforms, E. coli, and S. aureus, althoughlegislation does not establish any standard for this raw ma-terial. The difference in the counts of these contaminants inthe dry and rainy season was minimal (< 10 cfu.mL-1). Thelack of standardization in the salting of the cheese directlyaffects the salt content in EC (Pimentel Filho et al., 2005)and consequently its microbiota. S. aureus can be found inEC, since they are halophilic microorganisms and multiplyin food at salt concentrations of up to 15% (Le Loir et al.,2003). However, even with the presence of contaminatingmicroorganisms, the EC had a predominance of lactic acidbacteria (Galinari et al., 2011), which inhibits the growth ofpathogenic microorganisms (Mafu et al., 2011; Zmantar et

al., 2010).

The counts of mesophilic aerobes, coliforms, and E.

coli in milk were slightly higher during the rainy season,but the opposite occurred for S. aureus, which was higherduring the dry season (Table 2). In both periods of manu-facture, mesophilic aerobes and S. aureus counts wereabove the limits of legislation (> 5 Log cfu.mL-1 and> 2 Log cfu.mL-1, respectively), indicating deficiencies ingood manufacturing practices. Thus, the contamination ofthe water samples, milk, and EC are related to inadequatesanitary conditions found in the production units, since thehealth of the herd, affected by the possible presence of mas-titis, must be carefully controlled until the final stages ofproduction of cheese (when EC is collected) to improve thequality of the final product.

Microbiological analysis of the cheeses

For coliforms, E. coli, and S. aureus, the conditions ofcheese ripening were determined to promote the reduction

of these contaminants. From 29 days of ripening at roomtemperature, the counts of these microorganisms werelower than 10 cfu.g-1 for all samples (Figure 4). The oppo-site was found in cheeses ripened under refrigeration,which had higher microorganism counts, with results veri-fied through 64 days. Therefore, given the large differencebetween the two ripening conditions, the ANOVA for thecontaminants group considered the simple effect and the in-teractions of only two factors, ripening time and cheesemanufacture. For cheeses ripened at room temperature, theANOVA indicated a simple effect (p < 0.05) of the ripeningtime on all contaminants. For coliforms, there was an inter-action (p < 0.05) between the periods of ripening andcheese production, with the highest (p < 0.05) average oc-curring in the rainy season in the early ripening, which didnot occur again after the 8th day. In the rainy season, the firstsix days of ripening (between the 2nd and 8th day) were suf-ficient to reduce the initial counts of coliforms more than2 log. Furthermore, ANOVA of the cheese samples ripenedunder refrigeration indicated the simple effect of (p < 0.05)the period of manufacture on coliforms and S. aureus andripening time for all groups of contaminants. It was verifiedthat coliforms and S. aureus were higher (p < 0.05) duringthe rainy season when compared with counts in the dry sea-son. We confirmed that the sanitary conditions of produc-tion units should be more closely observed in the rainyseason, during which bacterial growth is favored because ofthe high humidity.

The ripening time caused a negative linear effect onthe count of coliforms, E. coli, and S. aureus, especially thecheeses ripened at room temperature, which had lowercounts for cheeses refrigerated throughout virtually wholeripening time (Figure 4). The initial count of contaminantswas high at the beginning of ripening, mainly in the rainyseason, when the temperature and RH were higher. Accord-ing to Masoud and Jakobsen (2005), the increase in temper-ature implies directly in higher microbial growth incheeses, whether endogenous, added, or contaminant.Some factors contributed to the high initial count, amongthem the low microbiological quality of milk and EC andpossible contamination caused by handlers and the intrinsiccharacteristics of cheeses, such as the high moisture contentand substrate availability, favoring microbial multiplica-

226 Martins et al.

Table 2 - Mean (Log cfu.mL-1) microbiological counts of endogenous culture and milk used in the manufacture of artisanal Minas cheese from the regionof Serro.

Period of manufacture Sample Aerobic mesophiles Coliforms E. coli S. aureus

Dry season Endogenous culture - 3.08 2.18 2.46

Milk 5.79 4.04 1.50 4.43

Rainy season Endogenous culture - 3.36 2.23 2.41

Milk 5.93 4.91 1.78 3.45

Lesgilation Milk < 5.00 - < 2.00 < 2.00

- not required by legislation.

tion. Since milk is not treated to remove contaminant mi-croorganisms, proper hygiene practices should be put inplace to reduce its initial count. An EC collected from con-taminated whey will cause recontamination throughout theproduction process, since it is used in cheese manufacturingon the subsequent days.

In the course of ripening, the initial count of all micro-bial groups was gradually reduced (p < 0.05) at a higherspeed for cheeses ripened at room temperature. We also ob-served that the cheeses manufactured in the rainy seasonshowed a severe reduction in counts within each conditionof ripening. Even under refrigeration, microbial counts also

Minimum ripening time of an artisanal cheese 227

Figure 4 - Counted estimates of total coliforms, E. coli and S. aureus in samples of artisanal Minas cheese from the region of Serro, ripened at room tem-perature and under refrigeration as a function of the ripening period (Rainy - filled line; Dry - dashed line). *Significant by Student’s t-test (p < 0.05).Dotted lines indicate the microbiological limits required by legislation of Minas Gerais after ripening.

decreased. However, the higher temperatures accelerate theripening (Cabezas et al., 2005; Moatsou et al., 2004; Öneret al., 2006; Psoni et al., 2003). The ripening at room tem-perature caused a decrease in moisture content, and there-fore, increasing the concentration of sodium chloride andreducing the Aw in cheeses would significantly reduce con-taminants. According to Beresford et al. (2001), beyondthese factors, the low redox potential in cheeses favors thereduction of undesirable microorganisms. The conditionsfor inhibition developed during ripening occurs primarilyby competition between the secondary microbiota andLAB, which can predominate in the cheese by lactic acidproduction, reduced sugar content and redox potential andby producing antimicrobial compounds (Caridi et al.,2003b; Manolopoulou et al., 2003). By presenting a lineartendency for reduction of the microbiological count duringripening, it is suggested that this process would be favoredif used milk with good microbiological quality to producecheeses.

The results of this study make it clear that the cheesesmade in the dry season, even those that present a smallerdaily rate of microbial reduction during ripening than in therainy season, quickly achieve the legislative standards(Minas Gerais, 2002, 2008). The cheeses ripened at roomtemperature showed microbiological counts within thesestandards for all the contaminants from 17 days of ripening.On the other hand, cheeses ripened under refrigerationachieved such standards at 33 days in the dry season and 63days in the rainy season. No samples of cheese showed thepresence of Listeria monocytogenes. However, samples ofone producer, ripened under refrigeration and manufac-tured in the rainy season, had Salmonella spp. in the firsttwo weeks (at 8 and 15 days ripening). However, after 22days, even under refrigeration, Salmonella was not de-tected, probably due to the accumulation of lactic acid inthe cheese and low pH values found in this sample (< 4.7).Several studies have demonstrated the antimicrobial activ-ity of LAB against pathogens such as Listeria

monocytogenes, S. aureus, E. coli, and Salmonella spp.(Caridi et al., 2003a; González et al., 2007; Macedo et al.,2004; Rodríguez et al., 2005). Higher temperatures can bean ally in cheese ripening by promoting the metabolism ofLAB and the accumulation of metabolites responsible forthe inhibition and elimination of contaminants. Therefore,the ripening of artisanal Minas cheese under refrigerationallows the placement of contaminated products on the mar-ket, representing risks to the consumer.

Analysis of staphylococcal enterotoxin

Although the initial counts of S. aureus in cheeseswere above those established by present legislation, entero-toxins were not detected in any sample. This results indi-cates the possible presence in the cheese of somemechanism inhibiting the expression of genes related totoxin production. Further studies in this area must be done

for conclusive statements, including the effects of theconditions (room temperature and refrigeration) and theripening time on the pre-formed toxin.

Conclusions

The effects of ripening at room temperature on thephysicochemical parameters of cheese directly influencedmicrobial behavior, causing a reduction in contaminantcounts. Thus, at room temperature, 17 days was the mini-mum ripening time for the cheeses to reach the microbio-logical standards required by law. In turn, the cheesesripened under refrigeration only reached such standards at33 days for cheeses manufactured in the dry season and 63days for those manufactured in the rainy season. Therefore,we suggest that the ripening of artisanal Minas cheese bedone at room temperature, since this condition shortens thetime needed to reach the microbiological quality that fallswithin the safety parameters required by legislation, andalso maintain the appearance and flavor characteristicsknown by producers and consumers who appreciate theartisanal Minas cheese.

Abbreviations

ANOVA, variance analysis; EC, endogenous culture;FDM, fat in dry matter; LAB, lactic acid bacteria; pH 4.6SN, pH 4.6 soluble nitrogen; RDI, ripening depth index;REI, ripening extension index; RH, relative humidity;TCA-SN, soluble nitrogen in 12% trichloracetic acid; TN,total nitrogen; TP, total protein; Aw, water activity.

Acknowledgments

This research was supported by CNPq. We would liketo thank the cheese makers for supplying the samples.

References

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